Mammalian sperm are not able to fertilize eggs immediately after ejaculation. They acquire fertilization capacity after residing in the female tract for a finite period of time in a process known as capacitation [24, 25]. Initially, capacitation was defined using fertilization as end-point. However, a variety of evidences suggest that the functional changes occurring in the sperm during capacitation are not one event, but a combination of sequential and concomitant processes. Some of these processes occur as soon as the sperm are released from the epididymis, others are slower and are activated only after sperm incubation for a certain period of time in conditions that support the sperm ability to fertilize the egg. These slow events are associated with changes in the motility pattern (e.g. hyperactivation) and with the acquisition of the sperm capacity to undergo an agonist-stimulated acrosome reaction (AR). Although both, fast and slow events are regulated by HCO3- and by a cAMP-dependent pathway, slower events are limited by the release of cholesterol from the sperm plasma membrane. Using the mouse as an experimental model, we have demonstrated that these last events are associated with a protein kinase A (PKA)-dependent increase in the tyrosine (tyr) phosphorylation of a subset of proteins [23, 26]. Simultaneously with our findings, Zeng et al. (1995) reported that capacitation is accompanied by hyperpolarization of the sperm plasma membrane ootential (Em). It has been hypothesized that hyperpolarization is necessary to drive Low Voltage Activated (LVA) Ca2+ T-channels (Cav3) from an inactive state to a closed state that can be activated by agonists such as the zona pellucida (ZP), triggering the AR. Despite these advances, little is known on how these molecular changes are combined to promote capacitation. As part of the first cycle of this grant, we have shown that HCO3-, Na+ and K+ are involved in the regulation of the sperm Em [21, 27, 28] and that Epithelial Na+ channels (ENaC) are present in sperm and play an important role in the regulation of the sperm resting Em [21]. The objective of this proposal is to understand how changes in cAMP, protein phosphorylation and hyperpolarization integrate to promote capacitation. Investigation of the crosstalk between cAMP and the changes in ion permeability is essential to understand the molecular basis of capacitation and to provide novel targets for pharmacological control of the fertilization process.